A common metaphor to describe the brain is that it is like a supercomputer. Consequently, current efforts at improving technologies for large-scale recording of brain function are primarily focused on measuring its electrical activity. However, unlike a supercomputer, the brain is an electrochemical machine. Superimposed upon its network of synaptic connections is a chemical connectome, a largely invisible network of neuromodulators, such as serotonin and neuropeptides (NPs), which exert a profound influence on brain function. Neuromodulators influence brain states that alter the computations performed by neural circuits, and are central to emotion, mood and affect. An understanding of neuromodulatory influences is therefore relevant to psychiatric disorders in humans. Without the ability to measure and manipulate the release of specific neuromodulators, our understanding of neuronal circuit function will be fundamentally incomplete. Nevertheless, a gap remains between our ability to monitor brain electrical activity, and our ability to monitor chemical activty with commensurate spatio-temporal resolution. Specifically, there is no method to visualize the release of specific NPs in the brain, at individual synapses. To fill this gap, we propose to develop a method for imaging the release of specific NPs at the level of nerve terminals, in vivo. The long-term goal is to develop new methods for visualizing, detecting and inhibiting NP release in vivo, and to apply these methods to understanding the dynamics of neuromodulation of specific, behaviorally relevant neural circuits. The overall goal of this proposal is to developa novel approach for time-resolved imaging of NP release from nerve terminals. The central objective of this proposal is to tag components of large dense core vesicles (LDCVs), and specific NPs, with pH-sensitive fluorescent proteins and to determine whether these reporters can be used to image neurosecretory granule release. Drosophila melanogaster provides a useful test-bed for this technology because of its genetic manipulability and sophisticated imaging methodologies. To achieve our objective, in Aim 1 we will fuse different pH-sensitive fluorescent reporters to the protein coding sequences of several LDCV-specific proteins and NPs, and generate transgenic flies. In Aim 2, we will use optogenetic activation of specific neuropeptidergic neurons containing these reporters in vivo, to determine whether activation-dependent increases in fluorescence can be detected, and distinguished from synaptic vesicle (SV) release. The contribution will be to determine the feasibility of the proposed approach, and to achieve a proof- of-principle application of the method. This contribution is significant, because it has the potential to create a transformative new technology with broad general applications. The contribution is innovative, because it combines expertise from cell biology, molecular genetics and neural circuit analysis to develop a novel methodology. The work proposed in this application will therefore benefit the field of neuroscience as a whole, and also enable studies of neural chemistry and circuitry whose dysfunction may underlie psychiatric disorders.